Salmonella typhimurium and many other species of Salmonella are major causes of morbidity and mortality in humans in the United States and throughout the world. Salmonellosis is acquired primarily through ingestion of contaminated meat, dairy, and poultry products. Thus, a better understanding of pathogenesis of Salmonella should be useful for prevention of disease through vaccine development, improved therapy and management of disease, and improved agricultural methods for food production. Plasmids have recently been associated with virulence of several species of Salmonella; however, little is known about the mechanism or the genetics of plasmid-mediated virulence. The objectives of the proposed research are therefore to identify at the molecular level mechanism(s) by which the plasmid contributes to the virulence of S. typhimurium. The first specific aim is to identify plasmid-encoded virulence factors and genes responsible for the spread S typhimurium beyond the small intestine of orally inoculated mice. The principal investigator has cloned and mutated virulence genes of the plasmid, and these results are the basis for the continued use of molecular plasmid, genetic analysis in dissecting plasmid- mediated virulence. Virulence plasmid-encoded proteins were not detectably expressed by S,. typhimurium growing in vitro, suggesting that virulence genes may be regulated so as to be expressed only in the animal host. Therefore, recently isolated Tn5 genes. The second specific aim is therefore to elucidate how plasmid-encoded virulence genes are regulated through construction of operon fusions and analysis of mRNA. Virulence plasmid-encoded proteins and their locations in S typhimurium cells will be examined as the third specific aim. These studies are a natural extension of efforts to further characterize virulence factors and their roles in eliciting protective immunity.